System useful for reporting protein-protein interactions in the bacterial periplasm

ABSTRACT

One aspect of the present invention relates to a reporter system for detection of protein-protein interactions in the periplasm of a prokaryotic host cell. The reporter system includes a first expression system which has a nucleic acid molecule encoding a first fragment of a reporter protein molecule, a nucleic acid molecule encoding a first signal sequence, and a nucleic acid molecule encoding a first member of a putative binding pair, where the nucleic acid molecule encoding the first fragment, the nucleic acid molecule encoding the first signal sequence, and the nucleic acid molecule encoding the first member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a first fusion protein. The reporter system also includes a second expression system which has a nucleic acid molecule encoding a second fragment of the reporter protein molecule, a nucleic acid molecule encoding a second signal sequence, and a nucleic acid molecule encoding a second member of the putative binding pair, where the nucleic acid molecule encoding the second fragment, the nucleic acid molecule encoding the second signal sequence, and the nucleic acid molecule encoding the second member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a second fusion protein, where, when expressed in a prokaryotic host cell, at least one of the first and the second fusion proteins are co-translationally transported to the periplasm where, when present, the first and second members of the putative binding pair bind together and the first and second fragments of the reporter protein molecule are reconstituted, thereby producing an active reporter protein. The reporter system may be used to carry out methods of identifying candidate compounds which bind to a target protein, identifying a candidate gene which modulates binding between a first protein and second protein, and identifying a candidate compound which modulates binding between a first protein and second protein.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/115,172, filed Nov. 17, 2008.

This invention was made with government support under grant number1R21CA132223 by the National Institutes of Health. The government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to a system useful for reportingprotein-protein interactions in the bacterial periplasm.

BACKGROUND OF THE INVENTION

Protein-protein interactions are key molecular events that integratemultiple gene products into functional complexes in virtually everycellular process. These cellular processes are mostly mediated bynon-covalently interacting multi-protein complexes and can include, forexample, transcription, translation, metabolic pathways or signaltransduction pathways. Because such interactions mediate numerousdisease states and biological mechanisms underlying the pathogenesis ofbacterial and viral infections, identification of protein-proteininteractions remains one of the most important challenges in thepost-genomics era. The most widely used is the yeast two-hybrid assay(Y2H), which was developed in 1989 (Fields et al., “A Novel GeneticSystem to Detect Protein-protein Interactions,” Nature 340; 245-246(1989)). Briefly, this assay comprises two proteins fused to a splityeast transcription factor (originally GAL4) that binds a promoterupstream of a reporter protein. If the proteins interact, the activityof the transcription factor is reconstituted and transcription of thereporter protein is upregulated, providing a signal.

The yeast 2-hybrid (Y2H) system has been the tool of choice forrevealing numerous protein-protein interactions that underly diverseprotein networks and complex protein machinery inside living cells. Y2Hassay has been used to generate protein interaction maps for humans(Rual J. F., et al., “Towards a Proteome-Scale Map of the HumanProtein-protein Interaction Network,” Nature 437:1173-1178 (2005)).Another important application of the Y2H methodology is the discovery ofdiagnostic and therapeutic proteins, whose mode of action ishigh-affinity binding to a target peptide or protein. For example,several groups have isolated antibody fragments that are readilyexpressed in the cytoplasm of cells where they bind specifically to adesired target (der Maur et al., “Direct in vivo Screening of IntrabodyLibraries Constructed on a Highly Stable Single-chain Framework,” J BiolChem 277:45075-45085 (2002), Visintin M., et al., “Selection ofAntibodies for Intracellular Function Using a Two-Hybrid in vivoSystem,” Proc Natl Acad Sci USA 96:11723-11728 (1999)), and in certaininstances ablate protein function (Tanaka T. et al., “Intrabodies Basedon Intracellular Capture Frameworks that Bind the RAS Protein with HighAffinity and Impair Oncogenic Transformation,” EMBO J. 22:1025-1035(2003), Tse E., et al., “Intracellular Antibody Capture Technology:Application to Selection of Intracellular Antibodies Recognising theBCR-ABL Oncogenic Protein,” J Mol Biol 31 7:85-94 (2002)). The yeasttwo-hybrid assay is highly versatile and is still widely used foranalysis of the complex interactions of eukaryotic cellular networks.However, it has several drawbacks, including in the fact that itrequires the nuclear environment of the eukaryotic yeast host, which maydiffer from the interaction environment of the proteins of interest.

The Y2H system was initially developed by using yeast as a hostorganism. Numerous bacterial (B)2H systems are now common laboratorytools and represent an experimental alternative with certain advantagesover the yeast-based systems (Hu J. C. et al., “Escherichia coli One-and Two-hybrid Systems for the Analysis and Identification ofProtein-protein Interactions,” Methods 20:80-94 (2000), Ladant D. etal., “Genetic Systems for Analyzing Protein-protein Interactions inBacteria,” Res Microbiol 151:711-720 (2000)). A number of thesebacterial approaches employ split activator/repressor proteins; thus,they are functionally analogous to the GAL4-based yeast system (Dove S.L. et al., “Activation of Prokaryotic Transcription Through ArbitraryProtein-protein Contacts,” Nature 386:627-630 (1997), Hu J. C., et al.,“Sequence Requirements for Coiled-coils: Analysis with lambdaRepressor-GCN4 Leucine Zipper Fusions,” Science 250:1400-1403 (1990),Joung J. K., et al., “A Bacterial Two-hybrid Selection System forStudying Protein-DNA and Protein-protein Interactions,” Proc Natl AcadSci USA 97:7382-7387 (2000)). Unfortunately, both Y2H and B2H GAL4-typeassays are prone to a high frequency of false positives that arise fromspurious transcriptional activation (Fields S., “High-throughputtwo-hybrid Analysis. The Promise and the Peril,” FEBS J 272: 5391-5399(2005)), and complicate the interpretation of interaction data. Asproof, a comparative assessment revealed that >50% of the data generatedusing Y2H were likely to be false positives (von Mering C., et al.,“Comparative Assessment of Large-scale Data Sets of Protein-proteinInteractions,” Nature 41 7:399-403 (2002)). To address this shortcoming,several groups have exploited oligomerization assisted reassembly ofsplit enzymes such as adenylate cyclase (Karimova G., et al., “ABacterial Two-hybrid System Based on a Reconstituted Signal TransductionPathway,” Proc Natl Acad Sci USA 95:5752-5756 (1998)), β-lactamase (Bla)(Wehrman T., et al., “Protein-protein Interactions Monitored inMammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,”Proc Natl Acad Sci USA 99:3469-3474 (2002)), and dihydrofolate reductase(Pelletier J. N., et al., “An in vivo Library-versus-library Selectionof Optimized Protein-protein Interactions,” Nat Biotech 17:683-690(1999), Pelletier J. N., et al., “Oligomerization Domain-directedReassembly of Active Dihydrofolate Reductase from Rationally DesignedFragments,” Proc Natl Acad Sci USA 95:12141-12146 (1998)), as well assplit fluorescent proteins (Ghosh I. et al., “Antiparallel LeucineZipper-Directed Protein Reassembly: Application to the Green FluorescentProtein,” J Am Chem Soc 122:5658-5659 (2000)). Alternatively, a numberof methodologies for detecting interacting proteins in bacteria havebeen developed that do not rely on interaction-induced complementationof protein fragments, but instead use phage display (Palzkill T. et al.,“Mapping Protein-ligand Interactions Using Whole Genome Phage DisplayLibraries,” Gene 221:79-83 (1998)), FRET (You X., et al., “IntracellularProtein Interaction Mapping with FRET Hybrids,” Proc Natl Acad Sci USA103:18458-18463 (2006)), and cytolocalization of GFP (Ding Z., et al.,“A Novel Cytology-based, Two-hybrid Screen for Bacteria Applied toProtein-protein Interaction Studies of a Type IV Secretion System,” JBacteriol 184:5572-5582 (2002)).

In recent years, an alternative to the yeast two-hybrid assay has arisenin the form of the protein complementation assay (PCA). This methodfuses the proteins of interest to a split reporter protein such as GFP(Cabantous, S. et al., “Recent Advances in GFP Folding Reporter andSplit-GFP Solubility Reporter Technologies. Application to Improving theFolding and Solubility of Recalcitrant Proteins from MycobacteriumTuberculosis,” J Struct Funct Genomics 6:113-119 (2005), Cabantous, S.et al., “In vivo and in vitro Protein Solubility Assays Using SplitGFP,” Nat Methods 3:845-54 (2006)), YFP (Bracha-Drori, K. et al.,“Detection of Protein-protein Interactions in Plants Using BimolecularFluorescence Complementation,” Plant J 40:419-427 (2004)), luciferase(Kim, S. B. et al., “High-throughput Sensing and Noninvasive Imaging ofProtein Nuclear Transport by Using Reconstitution of Split RenillaLuciferase,” Proc Natl Acad Sci USA 101:11542-11547 (2004)),dihydrofolate reductase (Remy, I. et al., “Detection of Protein-proteinInteractions Using a Simple Survival Protein-fragment ComplementationAssay Based on the Enzyme Dihydrofolate Reductase,” Nat Proto2:2120-2125 (2007)), or β-lactamase (Galarneau, A. et al.,“Beta-lactamase Protein Fragment Complementation Assays as in vivo andin vitro Sensors of Protein-protein Interactions,” Nat Biotechnol20:619-22 (2002), Wehrman, T. et al., “Protein-protein InteractionsMonitored in Mammalian Cells via Complementation of Beta-lactamaseEnzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002)). Usingprotein engineering techniques, split proteins whose individualfragments are inactive can be developed. Upon interaction of theproteins of interest, the split reporter protein regains its activity.Versions of a split β-lactamase (Bla) protein complementation assay formonitoring protein-protein interactions in mammalian cells have beendeveloped (Galarneau, A. et al., “Beta-lactamase Protein FragmentComplementation Assays as in vivo and in vitro Sensors ofProtein-protein Interactions,” Nat Biotechnol 20:619-22 (2002), Wehrman,T. et al., “Protein-protein Interactions Monitored in Mammalian Cellsvia Complementation of Beta-lactamase Enzyme Fragments,” Proc Natl AcadSci USA 99:3469-3474 (2002)). These versions however contain no signalsequence for protein transport, and thus the proteins were expressed inthe cytoplasm of E. coli. Following expression, the Bla (β-lactamase)activity was measured in vitro by nitrocefin colorimetric assay. Onelimitation of cytoplasmic expression is that cytoplasmic β-lactamase(Bla) is incapable of conferring resistance to ampicillin and thusgenetic selection is not possible. In one version, Wehrman et al.employed prototypical Sec signal peptides for delivery of the fragmentsinto the periplasm by the post-translational Sec export pathway(Wehrman, T. et al., “Protein-protein Interactions Monitored inMammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,”Proc Natl Acad Sci USA 99:3469-3474 (2002)). Following export, bothfragments localize to the periplasm and cells can be selected onampicillin. The limitation of this approach is that it usespost-translational Sec export signals. The proteins under investigationmay fold fully inside the cytoplasm thereby limiting their potentialtranslocation across the cytoplasmic membrane. They may also interact inthe cytoplasm prior to export and thus not be exported to the periplasm.A further limitation of this approach is the fact that only one pair ofsmall peptides was tested for interaction; whether split Bla(β-Lactamase) could be used to report the interactions between globularproteins was not demonstrated. Thus, to date there have been no reportsdetailing the use of split protein fragments for monitoringprotein-protein interactions in the bacterial periplasm.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a reporter system fordetection of protein-protein interactions in the periplasm of aprokaryotic host cell. The reporter system includes a first expressionsystem which has a nucleic acid molecule encoding a first fragment of areporter protein molecule, a nucleic acid molecule encoding a firstsignal sequence, and a nucleic acid molecule encoding a first member ofa putative binding pair, where the nucleic acid molecule encoding thefirst fragment, the nucleic acid molecule encoding the first signalsequence, and the nucleic acid molecule encoding the first member of theputative binding pair are operatively coupled to permit their expressionin a prokaryotic host cell as a first fusion protein. The reportersystem also includes a second expression system which has a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second member of the putative binding pair,where the nucleic acid molecule encoding the second fragment, thenucleic acid molecule encoding the second signal sequence, and thenucleic acid molecule encoding the second member of the putative bindingpair are operatively coupled to permit their expression in a prokaryotichost cell as a second fusion protein. When expressed in a prokaryotichost cell, at least one of the first and the second fusion proteins areco-translationally transported to the periplasm where, when present, thefirst and second members of the putative binding pair bind together andthe first and second fragments of the reporter protein molecule arereconstituted, thereby producing an active reporter protein.

Another aspect of the present invention relates to a method ofidentifying a candidate protein which binds a target protein. Thismethod includes providing a first expression system comprising a nucleicacid molecule encoding a first fragment of a reporter protein molecule,a nucleic acid molecule encoding first signal sequence, and a nucleicacid molecule encoding a target protein, where the nucleic acid moleculeencoding the first fragment, the nucleic acid molecule encoding thefirst signal sequence, and the nucleic acid molecule encoding the targetprotein are operatively coupled to permit their expression in aprokaryotic host cell as a first fusion protein. This method alsoincludes providing a second expression system comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a candidate protein, where the nucleic acidmolecule encoding the second fragment, the nucleic acid moleculeencoding the second signal sequence, and the nucleic acid moleculeencoding the candidate protein are operatively coupled to permit theirexpression in a prokaryotic host cell as a second fusion protein. Thismethod further includes transforming a prokaryotic host cell with thefirst expression system and the second expression system and culturingthe transformed prokaryotic host cell under conditions effective toexpress the first and the second fusion proteins and to transport thefirst fusion protein and the second fusion protein to the prokaryotichost cell's periplasm, with at least one of the first fusion protein orthe second fusion protein being co-translationally transported to theperiplasm. The prokaryotic host cells with reporter protein moleculeactivity are detected as those where binding between the candidateprotein and the target protein has occurred. The candidate protein isidentified as having the ability to bind to the target protein based onwhether the host cell has reporter protein activity.

Another aspect of the present invention is related to a method ofidentifying a candidate gene which modulates the binding between a firstprotein and a second protein. This method includes providing a firstexpression system comprising a nucleic acid molecule encoding firstfragment of a reporter protein molecule, a nucleic acid moleculeencoding a first signal sequence, and a nucleic acid molecule encoding afirst protein, where the nucleic acid molecule encoding the firstfragment, the nucleic acid molecule encoding the first signal sequence,and the nucleic acid molecule encoding the first protein are operativelycoupled to permit their expression in a prokaryotic host cell as a firstfusion protein. This method also includes providing a second expressionsystem for expressing the second protein comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second protein, where the nucleic acid moleculeencoding the second fragment, the nucleic acid molecule encoding thesecond signal sequence, and the nucleic acid molecule encoding thesecond protein are operatively coupled to permit their expression in aprokaryotic host cell as a second fusion protein. This method furtherinvolves providing a candidate gene in a form suitable for expression ina prokaryotic host cell, transforming the prokaryotic host cell with thefirst expression system, the second expression system, and the candidategene, and culturing the transformed prokaryotic host cell underconditions effective to, in the absence of the candidate gene, expressthe first fusion protein, the second fusion protein, and the proteinencoded by the candidate gene and transport the first fusion protein andthe second fusion protein to the prokaryotic host cell's periplasm withat least one of the first fusion protein or the second fusion proteinbeing co-translationally transported to the periplasm. Any reporteractivity in the transformed prokaryotic host cell is detected, andprokaryotic host cells, with reporter activity that is different thanthat achieved without transformation of the candidate gene areidentified, as containing a candidate gene which modulates bindingbetween the first and second proteins.

Another aspect of the present invention is related to a method ofidentifying a candidate compound which modulates binding between a firstprotein and a second protein. This method includes providing a firstexpression system comprising a nucleic acid molecule encoding firstfragment of a reporter protein molecule, a nucleic acid moleculeencoding a first signal sequence, and a nucleic acid molecule encoding afirst protein, where the nucleic acid molecule encoding the firstfragment, the nucleic acid molecule encoding the first signal sequence,and the nucleic acid molecule encoding the first protein are operativelycoupled to permit their expression in a prokaryotic host cell as a firstfusion protein. This method also includes providing a second expressionsystem for expressing the second protein comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second protein, where the nucleic acid encodingthe second fragment, the nucleic acid encoding the second signalsequence, and the nucleic acid encoding the second protein areoperatively coupled to permit their expression in a prokaryotic hostcell as a second fusion protein. This method further involves providinga candidate compound, transforming a host prokaryotic cell with thefirst expression system and the second expression system, and culturingthe transformed host prokaryotic cell under conditions effective toexpress the first and the second fusion proteins and transport the firstfusion protein and the second fusion protein to the prokaryotic hostcell's periplasm with at least one of the first fusion protein or thesecond fusion protein being co-translationally transported to theperiplasm. The candidate compound is contacted with the culturedprokaryotic host cell. The reporter activity is detected in thetransformed prokaryotic host cell, and candidate compounds contactingthe prokaryotic host cells, with reporter activity that is differentthan that achieved in transformed prokaryotic host cells not contactedwith the candidate compound are identified as modulating binding betweenthe first protein and the second protein.

While numerous assays exist for detecting protein interactions in thecytoplasm of living cells, the major advantage of the present inventionis that it can be used to assay for protein-protein interactions in theperiplasmic compartment of bacteria. Such an assay would be significantbecause of the importance of the bacterial periplasm in a large numberof biotechnological applications. This importance stems from severalunique features of the periplasm relative to other sub-cellularcompartments. For instance, proteins that require disulfide bonds forcorrect folding can only adopt a native conformation when localized inthe oxidizing environment of the bacterial periplasm (disulfide bonds donot normally form in the cytoplasm of bacteria). Examples ofdisulfide-bond containing proteins of biotechnological relevance includeproteins and protein fragments of the immunoglobulin family (e.g.,IgGs). The ability to detect protein interactions in the periplasm caneasily be combined with the ability to express full-length IgGs orfragments derived thereof (e.g., single-chain Fv (scFv)) in theperiplasm and opens the door to a cell-based selection system forisolating and engineering virtually all types of antibodies or otherimmune/non-immune binding proteins. Since the technology described aboveonly requires the antigen or domains of the antigen to be expressed inthe periplasm, this technology should enable the selection of antibodiesagainst both soluble and membrane proteins. Since many complex membraneproteins can be expressed in the inner membrane of E. coli (e.g., GPCRs,ion channels, efflux pumps, etc.), this technology should enableisolation of antibodies or other binding proteins that interact withexposed loops of membrane proteins. Such a feat is difficult orimpossible using traditional antibody selection methods (e.g., cellsurface display, phage display). More recently, the development ofN-linked glycosylation in bacteria opens the bacterial periplasm as acompartment for attaching N-glycans to specific residues in recombinantproteins. Another advantage of the periplasm is that isolation ofrecombinant proteins from this compartment is greatly simplifiedcompared to isolation from the more crowded cytoplasm. To accomplishthis while also overcoming previous limitations of split β-lactamasesystems in E. coli, a preferred embodiment of the present invention isco-expression of β-lactamase fragment fusions that are engineered withco-translational export signals (e.g., signal peptides from the E. coliDsbA protein). The advantage of such export signals is that at least oneor both fragments of the reporter protein, and the interacting proteins(members of the putative binding pair) to which they are fused, arelocalized directly into the periplasm with little or no residence timein the cytoplasm. As a result, premature interaction between theproteins under investigation is effectively eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing protein 1 to protein 2interactions complementing the two split fragments of β-lactamase. Thisarrangement enables the enzyme to hydrolyze ampicillin and cells to growin the presence of the antibiotic.

FIGS. 2A-D show the protein complementation of interacting domains. FIG.2A shows a schematic drawing of the two-plasmid system comprising genesof interest fused to α and ω β-lactamase fragments. Fos and Jun are usedhere as examples, and putative binding partners of interest wouldreplace Fos and Jun in their respective constructs. FIG. 2B shows spotplating of Fos and Jun leucine zippers on 50 μg/ml Amp. GCN4, anotherleucine zipper, but one that does not interact with Fos, is used as anegative control. FIG. 2C shows spot plating as in FIG. 2B of antibodyfragment scFv-GCN4 and its antigen, the GCN4 leucine zipper. GCN4-PP isa double point mutant to wild-type GCN4 that reduces homodimerization ofthe leucine zippers. Jun is used as a negative control. FIG. 2D showsspot plating of scFv D10, with affinity to phage protein Gpd. Jun andGCN4 used as negative controls.

FIGS. 3A-B show the interaction of Fos and Jun variants. FIG. 3A showsspot plating as in FIG. 2B of Fos/Jun variants. Notations such as L3Vrefer replacement of leucine 3 with valine, not necessarily residue 3.Similar growth for all variants was confirmed on Cm/Kan plates. FIG. 3Bshows MIC (blue) and MBC (red) measurements of Fos and Jun variants asshown in FIG. 3A.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a reporter system (alsodescribed herein as protein complementation system) which can be used todetect, protein-protein interactions. FIG. 1 shows one embodiment of thepresent invention. FIG. 1 shows protein 1 and protein 2 attached to twofragments of Bla. The proteins interact with one another and lead tocomplementation of the Bla fragments thus reconstituting the reporteractivity. Bacterial cells transformed with this system will survive onampicillin media. The reconstituted reporter activity of Bla leads tohydrolysis of ampicillin and enables the bacterial cells to survive onampicillin media.

In one aspect, the present invention relates to a reporter system fordetection of protein-protein interactions in the periplasm of aprokaryotic host cell. The reporter system includes a first expressionsystem which has a nucleic acid molecule encoding a first fragment of areporter protein molecule, a nucleic acid molecule encoding a firstsignal sequence, and a nucleic acid molecule encoding a first member ofa putative binding pair, where the nucleic acid molecule encoding thefirst fragment, the nucleic acid molecule encoding the first signalsequence, and the nucleic acid molecule encoding the first member of theputative binding pair are operatively coupled to permit their expressionin a prokaryotic host cell as a first fusion protein. The reportersystem also includes a second expression system which has a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second member of the putative binding pair,where the nucleic acid molecule encoding the second fragment, thenucleic acid molecule encoding the second signal sequence, and thenucleic acid molecule encoding the second member of the putative bindingpair are operatively coupled to permit their expression in a prokaryotichost cell as a second fusion protein. When expressed in a prokaryotichost cell, at least one of the first and the second fusion proteins areco-translationally transported to the periplasm where, when present, thefirst and second members of the putative binding pair bind together andthe first and second fragments of the reporter protein molecule arereconstituted, thereby producing an active reporter protein.

The reporter system of the present invention is useful in detectinginteraction between, for example, a known first member of a putativebinding pair and a second member, which was previously not known to bindthe first member. The method detects the interaction of the first memberwith the second member by bringing into close proximity members of afragment pair of a reporter protein, such that the reporter protein isreassembled to its original functionality or enzymatic activity. Thefragments of the reporter protein of the present invention can interactto produce a detectable signal such as a visible phenotypic change orantibiotic resistance. This system should enable, for example, theidentification of molecules and/or genes that promote or inhibit keyprotein interactions, existing in a range of cell types, phyla andspecies, via high-throughput screens.

As used herein, a “reporter protein” refers to a protein which can beseparated into two or more individual protein fragments, where thereporter protein fragments are capable of associating with each other togenerate a detectable signal, or are capable of associating with eachother and one or more additional substances to generate a detectablesignal, and which do not individually generate the detectable signal.The functional fragments of a reporter protein of interest can beidentified by methods well known in the art. For example, it can involvepreparing a multiplicity of fragment pair members with breaks within asolvent exposed loop or a flexible loop defined by tertiary or secondarystructure analysis to obtain a fragment pair library. The fragment pairmembers can be, for example, expressed in a multiplicity of host cells,and the host cells exhibiting a directly detectable signal associatedwith the reporter protein of interest can be isolated as indicative ofcontaining fragment pair members that functionally reconstitute thereporter protein. Plasmids containing expression systems coding for thefragment pair members can then be sequenced to identify functionalfragment pairs of the reporter protein. Other methods of breakingproteins into fragments are known in the art and can be applied to thepresent invention as long as the reporter protein functions in a mannerdescribed supra.

In a preferred embodiment the reporter system comprises the antibioticresistance enzyme, β-lactamase as a reporter protein molecule. However,fragment pairs of other enzymes that provide for antibiotic resistanceare also included in the present invention, including, for example,aminoglycoside phosphotransferases, particularly neomycinphosphotransferase, chloramphenicol acetyl transferase, and tetracyclineresistance protein (Backman et al., “Tetracycline Resistance Determinedby pBR322 is Mediated by One Polypeptide,” Gene 26:197 (1983), which ishereby incorporated by reference in its entirety). Other proteins thatcan directly elicit a visible phenotypic change such as a color changeor fluorescence emission also are applicable to the present invention.Examples of such proteins include β-galactosidase and green fluorescentprotein (GFP) or other related fluorescent proteins.

The reporter protein molecule can be selected from a group consisting ofa monomeric protein, a multimeric protein, a monomeric receptor, amultimeric receptor, a multimeric biomolecular complex, adenylatecyclase, alkaline phosphatase, β-lactamase, cellulase, chloramphenicolacetyl transferase (CAT), disulfide bond oxidase A (DsbA), maltosebinding protein (MBP), methyltransferase, dihydrofolate reductase(DHFR), luciferase, thymidylate synthase, thymidine kinase, Trp1N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and allfluorescent proteins including green fluorescent protein (GFP), bluefluorescent protein (BFP), cyan fluorescent protein (CFP), redfluorescent protein (RFP), yellow fluorescent protein (YFP), monomericRFP (mRFP), mCherry, mOrange, mBanana, mStrawberry, mHoneydew, tdTomato,and mTangerine.

The β-lactamase of E. coli is a 286 amino acid (including the signalsequence) product of the ampicillin resistance gene of plasmid pBR322(Sutcliffe J G., “Nucleotide Sequence of the Ampicillin Resistance Geneof Escherichia coli Plasmid pBR322,” Proc Natl Acad Sci USA 75(8):3737-41 (1978), which is hereby incorporated by reference in itsentirety) and has the following amino acid sequence (Genbank IDAAB59737.1) (SEQ ID NO: 1):

  1 msiqhfrval ipffaafclp vfahpetlvk vkdaedqlga rvgyieldln 51 sgkilesfrp eerfpmmstf kvllcgavls rvdagqeqlg rrihysqndl101 veyspvtekh ltdgmtvrel csaaitmsdn taanllltti ggpkeltafl151 hnmgdhvtrl drwepelnea ipnderdttm paamattlrk lltgelltla201 srqqlidwme adkvagpllr salpagwfia dksgagergs rgiiaalgpd251 gkpsrivviy ttgsqatmde rnrqiaeiga slikhw

The β-lactamase reporter protein molecule may be split into a firstfragment and a second fragment. The first fragment of the β-lactamasereporter protein can comprise an α fragment with residues 1-196 of theβ-lactamase, where the α fragment has the following amino acid sequence(SEQ ID NO: 2):

  1 msiqhfrval ipffaafclp vfahpetlvk vkdaedqlga rvgyieldln 51 sgkilesfrp eerfpmmstf kvllcgavls rvdagqeqlg rrihysqndl101 veyspvtekh ltdgmtvrel csaaitmsdn taanllltti ggpkeltafl151 hnmgdhvtrl drwepelnea ipnderdttm paamattlrk lltgelThe second fragment of the β-lactamase reporter protein comprises ωfragment with residues 197-286 of the β-lactamase, where the ω fragmenthas the following amino acid sequence (SEQ ID NO: 3):

 1 ltlasrqqli dwmeadkvag pllrsalpag wfiadksgag ergsrgiiaa51 lgpdgkpsri vviyttgsqa tmdernrqia eigaslikhwFor example, the β-lactamase α and ω fragments can be used in thepresent invention to complement and produce selectable activity in thebacterial periplasm in a manner that is dependent on specificinteraction between the first member of putative binding pair) and thesecond member of the putative binding pair fused to the β-lactamasefragments. This β-lactamase based protein-protein interaction reportersystem can undergo co-translational secretory translocation across thebacterial inner membrane into the periplasm, and therefore can reliablydetect interactions between and among proteins.

The members of a putative binding pair which can be assayed for theirbinding affinity with each other, using the methods of the presentinvention, include any molecules capable of a binding interaction. Thebinding interaction between the two or more binding members may beeither direct or in the form of a complex with one or more additionalbinding species, such as charged ions or molecules, ligands, ormacromolecules. Putative binding partners, or putative binding moieties,according to the present invention, can include molecules which do notnormally interact with each other, but which interact with a thirdmolecule such that, in the presence of the third molecule, the putativebinding partners are brought together. Thus, substances which influencean interaction between putative binding partners include those whichstimulate a weak interaction between putative binding partners, as wellas one or more molecules which mediate interaction between moleculeswhich do not normally interact with each other. In addition, substanceswhich influence an interaction between putative binding partners caninclude those which directly or indirectly affect an upstream eventwhich results in association between the putative binding partners. Forexample, phosphorylation of one of the putative binding partners canendow it with the capacity to associate with another of the putativebinding partners.

Exemplary putative binding pairs include membrane protein-solublebinding protein pair, a membrane protein-membrane protein binding pair,a biotin-avidin binding pair, ligand-receptor binding pair, andantibody-antigen binding pair.

In an antigen-antibody pair, for example, the antibody can be amonoclonal antibody, bispecific antibody, single-chain antibody (scAb),single-chain Fv fragment (scFv), scFv₂, dsFv, scFv-Fc, Fab, F(ab′)₂,F(ab)₃, V_(L), diabody, single domain antibody, camelid antibody,triabody, tetrabody, minibody, one-armed antibody, and immunoglobulin(Ig), IgM, IgE, IgA, IgD, IgG, IgG-ΔC_(H)2.

The ligand-receptor pairs of the present invention can include, forexample, the following receptors Fc receptors (FcR), single-chain MHC,or single-chain T-cell receptor (sc-TCR). Useful ligands are, forexample, monotopic membrane proteins, polytopic membrane proteins,transmembrane proteins, G protein-coupled receptors (GPCRs), ionchannels, members of the SNARE protein family, integrin adhesionreceptor, multi-drug efflux transporters.

Other exemplary proteins include members of the signal transductionpathway, cell surface receptors, proteins regulating apoptosis, proteinsthat regulate progression of the cell-cycle, proteins involved in thedevelopment of tumors, transcriptional-regulatory proteins, translationregulatory proteins, proteins that affect cell interactions, celladhesion molecules, proteins that participate in the folding of otherproteins, and proteins involved in targeting to intracellularcompartments.

Members of signal transduction pathways include protein hormones andcytokines. Cytokines include those involved in signal transduction, suchas interferons, chemokines, and hematopoietic growth factors. Otherexemplary proteins include interleukins, lymphotoxin, transforminggrowth factors-a and 13, and macrophage and granulocyte colonystimulating factors. Other proteins include intracellular enzymes suchas protein kinases, phosphatases and synthases.

Exemplary proteins involved in apoptosis include tumor necrosis factor(TNF), Fos ligand, interleukin-113 converting enzyme (ICE) proteases,and TNF-related apoptosis-inducing ligand (TRAIL). Proteins involved inthe cell cycle include deoxyribonucleic acid (DNA) polymerases,proliferating cell nuclear antigen, telomerase, cyclins, cyclindependent kinases, tumor suppressors and phosphatases. Proteins involvedin transcription and translation include ribonucleic acid (RNA)polymerases, transcription factors, enhancer-binding proteins andribosomal proteins. Proteins involved in cellular interactions such ascell-to-cell signaling include receptor proteins, and peptide hormonesor their enhancing or inhibitory mimics.

The reporter protein fragments and one or more members of the putativebinding pair are generally linked either directly or via a linker, andare generally linked by a covalent linkage. For example, when thereporter protein fragment and the members of the putative binding pairare proteins, they may be linked by methods known in the art for linkingpeptides.

The fragment members of reporter proteins also may include a flexiblepolypeptide linker separating the fragment of reporter protein from themember of the putative binding pair and allowing for their independentfolding. The linker is optimally 15 amino acids or 60 Å in length (˜4 Åper residue) but may be as long as 30 amino acids but preferably notmore than 20 amino acids in length. It may be as short as 3 amino acidsin length, but more preferably is at least 6 amino acids in length. Toensure flexibility and to avoid introducing steric hindrance that mayinterfere with the independent folding of the fragment domain ofreporter protein and the members of the putative binding pair, thelinker should be comprised of small, preferably neutral residues such asGly, Ala, and Val, but also may include polar residues that haveheteroatoms such as Ser and Met, and may also contain charged residues.

In one embodiment, the β-lactamase reporter fragments are capable ofbinding to one another to form an enzymatically active complex that iscapable of catalyzing the conversion of a substrate to a product whichis detectable, either directly or indirectly. In one embodiment, theβ-lactamase reporter system can include two or more components, in afusion protein, where the fusion proteins each comprise a putativebinding protein fused to a low affinity β-lactamase reporter fragment.Thus, nucleic acids encoding the fusion proteins can be constructed,introduced into cells and expressed in cells. Alternatively, the boundβ-lactamase reporter units or bound binding moieties can be detected byselecting the prokaryotic host cells expressing the fusion proteins ofthe present invention on a selection media which includes an antibioticsuch as ampicillin.

A variety of cell-based assays can be conducted using the cellscontaining the fusion gene constructs. Binding of the putative bindingmoieties on the fusion proteins expressed in the cells can be confirmedby detecting the signal produced by the reporter fragments undergoingcomplementation. This signal could be, for example, a fluorescent signalwhich is regenerated after the reporter fragments undergocomplementation.

In one embodiment of the invention, prokaryotic cells in which a thereporter fragments undergo complementation can be detected and isolatedby flow cytometry or fluorescence-activated cell sorting (FACS). Methodsfor flow cytometry and FACS are well-known in the art (Nolan et al.,“Fluorescence-activated Cell Analysis and Sorting of Viable MammalianCells Based on beta-D-galactosidase Activity After Transduction ofEscherichia coli lacZ,” Proc. Natl. Acad. Sci. USA 85:2603-2607 (1988);Webster et al., “Isolation of human myoblasts with thefluorescence-activated cell sorter,” Exp. Cell Research, 174:252-265(1988), which are hereby incorporated by reference in their entirety).In this way, clones of cells in which binding occurs can be isolated andpropagated for further study.

Binding of the protein molecules of the present invention will dependupon factors such as pH, ionic strength, concentration of components ofthe assay, and temperature. In the binding assays using reporter systemsdescribed herein, the binding affinity of the first member of theputative binding pair and the second member of the putative binding pairshould be strong enough to permit binding between the reporter proteinfragments. In a preferred embodiment, the binding affinity of the firstmember of the putative binding pair and the second member of theputative binding pair should be stronger than the binding affinity ofthe first fragment and the second fragment of the reporter protein. Whencombining the first and second fusion proteins, the reconstitution ofthe first and second fragments into the reporter protein requires theinteraction between the first and second members of the putative bindingpair. Bound members of the putative binding pair are identified byexpressing a functionally reconstituted reporter protein, and then thenucleotide sequences encoding for bound members of the putative bindingpair are characterized by methods including electrophoresis, polymerasechain reaction (PCR), nucleotide and amino acid sequencing and the like.

In one embodiment, either one or both the first and second members ofthe putative binding pair can be a member of a library. The members ofthe putative binding pair can be parts of libraries that are constructedfrom cDNA, but may also be constructed from, for example, synthetic DNA,RNA and genomic DNA.

A large number of proteins synthesized in prokaryotes are translocated(transported) partially or fully to the outside of the cytoplasm by asecretory pathway. The first step of translocation involves insertioninto and translocation across the cytoplasmic membrane. In gram-positivebacteria, fully translocated proteins are released into the externalmilieu whereas in gram negative bacteria, the proteins are translocatedand released into the periplasm or are integrated into or transportedacross the outer membrane. The secreted proteins usually contain asecretory signal sequence (also referred to as a signal sequence or asignal peptide) that generally, but not always, contains a stretch of atleast around 10 hydrophobic amino acid residues. A detailed review ofthe general secretory pathways (GSP) in prokaryotic bacteria is providedby Pugsley A. P., “The Complete General Secretory Pathway inGram-negative Bacteria,” Microbiological Reviews 57:50-108 (1993), whichis hereby incorporated by reference in its entirety.

The proteins of the present invention are transported to the prokaryotichost cell's periplasm. This can be done, for example, by targeting thegeneral secretory pathway (GSP) of the prokaryotic cells to transportthe proteins across the cytoplasmic membrane. These secretory pathways(also known as translocation pathways or transport pathways) include,for example the Sec pathway, the SRP pathway and the Tat pathway.

For the purposes of the present invention, it is preferable that theproteins are maintained in an translocation competent conformation orfold. Proteins that are exported from the cytosol by the transportationpathways of prokaryotes have certain features that promote efficienttranslocation across membranes. These features generally, but notalways, include an amino-terminal hydrophobic signal sequence that iscleaved during the translocation process. Furthermore, some feature ofthe protein or of the export process must ensure that the protein doesnot fold such that its export is prevented. This is usually done inprokaryotic cells in several different ways. The proteins may betranslocated across a membrane simultaneously with translation(co-translationally) of the protein, thus ensuring that not even itssecondary structures are formed in the cytoplasm. If the proteins arenot co-translationally translocated then chaperones or antifoldingfactors may prevent folding in the cytoplasm thereby maintaining theexport competent conformation (Randall et al., “SecB: A Chaperone fromEscherichia coli,” Methods Enzymol. 290:444-459 (1998), which is herebyincorporated by reference in its entirety). In some cases, signalsequences act as intrapolypeptide chaperones to prevent rapid folding(Liu et al., “Physiological Role During Export for the Retardation ofFolding by the Leader Peptide of Maltose-binding Protein,” Proc NatlAcad Sci USA 86:9213-9217, which is hereby incorporated by reference inits entirety). Also the proteins that are exported post-translationallymay have features in their final structures (for example, disulfidebonds) that do not form in the environment of the cytoplasm so that theproteins cannot attain their final folded conformations in the cytoplasm(Derman et al., “Escherichia coli Alkaline Phosphatase Fails to AcquireDisulfide Bonds When Retained in the Cytoplasm,” J. Bacteriol173:7719-7722, which is hereby incorporated by reference in itsentirety). This is notable because antibodies are stabilized bydisulfide bonds and the oxidizing environment of the bacterial periplasmis conducive for the formation of disulfide bonds.

The proteins of the present invention comprise a signal sequence (alsoknown in the art as the targeting sequence or signal peptide) which,when expressed in a suitable host cell, directs the transport across orthrough the membrane. In accordance with the present invention, thefusion proteins are transported to the bacterial periplasm of gramnegative bacteria. Generally, but not always, the signal peptides arelocated at the amino termini of fusion proteins. The signal peptide istypically cleaved following its entry into the periplasm. As notedabove, at least one of the first or second fusion proteins isco-translationally transferred to periplasm of the prokaryotic hostcell.

The fusion protein can have signal sequences targeting the Sectranslocation pathways (Pugsley A. P., “The Complete General SecretoryPathway in Gram-negative Bacteria,” Microbiological Reviews 57:50-108(1993), Kumamoto C. A., “Molecular Chaperones and Protein Translocationacross the Escherichia coli inner membrane,” Mol. Microbiol. 5:19-22(1991), which are hereby incorporated by reference in their entirety).The Sec signal sequences can be selected from the group consistingssAppA, ssBla, ssClyA, ssLep, ssMalE, ssOmpA, ssOmpT, ssOmpX, ssPelB(Erwinia chrysanthemi), ssPhoA, ssRbsB, and ssYebF.

One component of the bacterial general secretory pathway that can beused in the present invention is the signal recognition pathway (SRP)(Pugsley A. P., “The Complete General Secretory Pathway in Gram-negativeBacteria,” Microbiological Reviews 57:50-108 (1993), Valent Q. A.,“Signal Recognition Particle Mediated Protein Targeting in Escherichiacoli,” Antonie van Leeuwenhoek 79:17-31 (2001), Koch et al., “SignalRecognition Particle-dependent Protein Targeting, Universal to AllKingdoms of Life,” Rev Physiol Biochem Pharmacol. 146:55-94 (2003),which are hereby incorporated by reference in its entirety). Theprokaryotic SRP is composed of one protein (Ffh) and a 4.5S RNA and ismainly involved in co-translational assembly of intergral membraneproteins (Luirink J. et al., “Mammalian and Escherichia coli SignalRecognition Particles,” Mol. Microbiol. 11:9-13 (1994), which is herebyincorporated by reference in its entirety). The SRP signal sequencesthat can be used for the purposes of the present invention can beselected from the group consisting ssArtI, ssDsbA, ssFlgA, ssLivJ,ssSfmC, ssSTII, ssTolB, ssTorT, ssYraP and ssYraI. The major advantageof using the SRP pathways is that it can transport the proteinco-translationally across the cytoplasmic membrane. This is a uniquefeature and ensures that the protein has little or no residence time inthe cytoplasm. Using the SRP pathways also overcomes the problems anddifficulties associated with translocating cytoplasmic proteins acrossthe cytoplasmic membrane which may be due to folding of the protein intoa conformation that has difficulty during translocation.

The reporter system of the present invention can include signalsequences which are related to the twin-arginine protein transportpathway (Tat pathway) (Sargent et al., “Pathfinders and Trailblazers: AProkaryotic Targeting System for Transport of Folded Proteins,” FEMSMicrobiol Lett 254:198-207 (2006), which is hereby incorporated byreference in its entirety). The Tat pathway for translocation is foundin the membranes of many bacteria and its most remarkable feature is itsability to transport prefolded, and often oligomeric proteins acrossionically sealed membranes (Berks et al., “The Tat Protein TranslocationPathway and Its Role in Microbial Physiology,” Adv Microb Physiol47:187-254 (2003), which is hereby incorporated by reference in itsentirety). The fusion proteins of the present invention can includedistinctive N-terminal signal peptides that bear a common amino acidsequence motif. This so-called “twin-arginine” motif has a consensussequence which includes Arginine residues and are essential forefficient protein translocation using the Tat pathway. The Tat pathwayhas an intrinsic “quality control” activity that prevents transport ofunfolded polypetides (DeLisa et al., “Folding Quality Control in theExport of Proteins by the Bacterial Twin Arginine TranslocationPathway,” Proc Natl Acad Sci USA 100: 6115-6120 (2003) which is herebyincorporated by reference in its entirety). A major advantage of usingthe Tat pathways in the present invention, is its innate ability toreject immature or incorrectly assembled protein and actively select forfolded substrates. The reporter system and the fusion proteins of thepresent invention can have signal peptides selected from the groupconsisting Tat signal sequences which include ssFdnG, ssFdoG, ssNapG,ssNrfC, ssHyaA, ssYnfE, ssWcaM, ssTorA, ssNapA, ssYagT, ssYcbK, ssDmsA,ssYdhX, ssYahJ, ssYedY, ssCueO, ssSufI, ssYcdB, ssTorZ, ssHybA, ssYnfF,ssHybO, ssAmiC, ssAmiA, ssYfhG, ssMdoD, ssFhuD, ssYaeI, and ssYcdO.

In one embodiment the reporter system of the present invention is suchthat both of the first and the second fusion proteins are transportedco-translationally to the prokaryotic host cell's periplasm.

Fusion proteins of the present invention, comprise a single continuouslinear polymer of amino acids which comprise the full or partialsequence of two or more distinct proteins. The construction of fusionproteins is well-known in the art. Two or more amino acids sequences maybe joined chemically, for instance, through the intermediacy of acrosslinking agent. In a preferred embodiment, a fusion protein isgenerated by expression of a fusion gene construct in a host cell.Fusion gene constructs generally also contain replication origins activein host cells and one or more selectable markers encoding, for example,drug or antibiotic resistance. The present invention is also directed toplasmids containing expression system constructed to express fusionproteins, described supra, of the present invention. The expressionsystems for the fusion protein will comprise operably linked nucleicacid components in the direction of transcription nucleotide sequencesencoding for (i) a promoter or other regulatory sequence functional in aprokaryotic host cell, (ii) a fragment of a reporter protein thatprovides for a directly selectable phenotype, (iii) a member of theputative binding pair, or a target protein or a candidate protein, (iv)an optional flexible polypeptide linker connecting the reporter proteinfragment to the member of the putative binding pair and (v) a signalpeptide or signal sequence. The invention is also concerned withprokaryotic host cells that contain plasmids having the sequences of theabove-described expression systems.

Different fusion gene constructs encoding unique fusion proteins may bepresent on separate nucleic acid molecules or on the same nucleic acidmolecule. Inclusion of different fusion gene constructs on the samenucleic acid molecule is advantageous, in that uptake of only a singlespecies of nucleic acid by a host cell is sufficient to introducesequences encoding both putative binding partners into the host cell. Bycontrast, when different fusion constructs are present on differentnucleic acid molecules, both nucleic acid molecules must be taken up bya particular host cell for the assay to be functional. Thus, problems ofcell mosaicism are avoided when both fusion gene constructs are includedon the same nucleic acid molecule.

Once the fusion protein is identified, the nucleic acid constructencoding the protein is inserted into an expression system to which themolecule is heterologous. The heterologous nucleic acid molecule isinserted into the expression system or vector in proper sense (5′→3′)orientation relative to the promoter and any other 5′ regulatorymolecules, and correct reading frame. The preparation of the nucleicacid constructs can be carried out using standard cloning methods wellknown in the art as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory Press, Cold Springs Harbor,N.Y. (1989), which is hereby incorporated by reference in its entirety.U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference in its entirety, also describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase.

A variety of prokaryotic expression systems can be used to express thefusion proteins of the present invention. Expression vectors can beconstructed which contain a promoter to direct transcription, a ribosomebinding site, and a transcriptional terminator. Examples of regulatoryregions suitable for this purpose in E. coli are the promoter andoperator region of the E. coli tryptophan biosynthetic pathway (Yanofskyet al., “Repression is Relieved Before Attenuation in the trp Operon ofEscherichia coli as Tryptophan Starvation Becomes IncreasinglySevere,”J. Bacteria. 158:1018-1024 (1984), which is hereby incorporatedby reference in its entirety) and the leftward promoter of phage lambda(N) (Herskowitz et al., “The Lysis-lysogeny Decision of Phage LambdaExplicit Programming and Responsiveness,” Ann. Rev. Genet., 14:399-445(1980), which is incorporated by reference in its entirety). Vectorsused for expressing foreign genes in bacterial hosts generally willcontain a sequence for a promoter which functions in the host cell.Plasmids useful for transforming bacteria include pBR322 (Bolivar etal., “Construction and Characterization of New Cloning Vehicles II. AMultipurpose Cloning System,” Gene 2:95-113 (1977), which is herebyincorporated by reference in its entirety), the pUC plasmids (Messing,“New M13 Vectors for Cloning,” Meth. Enzymol. 101:20-77 (1983), Vieiraet al., “New pUC-derived Cloning Vectors with Different SelectableMarkers and DNA Replication Origins,” Gene 19:259-268 (1982) which arehereby incorporated by reference in their entirety), and derivativesthereof. Plasmids may contain both viral and bacterial elements. Methodsfor the recovery of the proteins in biologically active form arediscussed in U.S. Pat. No. 4,966,963 to Patroni and U.S. Pat. No.4,999,422 to Galliher, which are incorporated herein by reference intheir entirety. See Sambrook, et al (In Molecular Cloning: A LaboratoryManual, 2^(nd) Ed., 1989, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, which is hereby incorporated by reference in itsentirety) for a description of other prokaryotic expression systems andmethods used for protein expression. Suitable expression vectors includethose which contain replicon and control sequences that are derived fromspecies compatible with the host cell. For example, if E. coli is usedas a host cell, plasmids such as pUC19, pUC18 or pBR322 may be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and, subsequently, the amount of fusion protein that isexpressed within the host cell. Transcription of DNA is dependent uponthe presence of a promoter, which is a DNA sequence that directs thebinding of RNA polymerase, and thereby promotes mRNA synthesis.Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters to obtain a high level oftranscription and, hence, expression and surface display. Depending uponthe host system utilized, any one of a number of suitable promoters maybe used. For instance, when using E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV 5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Translation of mRNA in prokaryotes depends upon the presence of theproper prokaryotic signals, which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference in its entirety.

Appropriate host cells for application of the present invention areprokaryotic host cells, such as bacterial cells. In a preferredembodiment, the host cell is a gram-negative bacteria. Host cellssuitable for expressing the proteins of the present invention includeany one of the more commonly available gram-negative bacteria. Suitablemicroorganisms include Pseudomonas aeruginosa, Escherichia coli,Salmonella gastroenteritis (typhimirium), S. typhi, S. enteriditis,Shigella flexneri, S. sonnie, S. dysenteriae, Neisseria gonorrhoeae, N.meningitides, Haemophilus influenzae H. pleuropneumoniae, Pasteurellahaemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum,T. denticola, T. orales, Borrelia burgdorferi, Borrelia spp. Leptospirainterrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P.mirabilis, Rickettsia prowazeki, R. typhi, R. richettsii, Porphyromonas(Bacteriodes) gingivalis, Chlamydia psittaci, C. pneumoniae, C.trachomatis, Campylobacter jejuni, C. intermedis, C. fetus, Helicobacterpylori, Francisella tularenisis, Vibrio cholerae, Vibrioparahaemolyticus, Bordetella pertussis, Burkholderie pseudomallei,Brucella abortus, B. susi, B. melitensis, B. canis, Spirillum minus,Pseudomonas mallei, Aeromonas hydrophile, A. salmonicida, and Yersiniapestis. Methods for transforming/transfecting host cells with expressionvectors are well-known in the art and depend on the host system selectedas described in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Laboratory Press, Cold Springs Harbor, N.Y. (1989), whichis hereby incorporated by reference in its entirety.

As will be apparent to one of skill in the art, the present inventionallows for a broad range of studies of protein-protein and other typesof multi-protein interactions to be carried out quantitatively orqualitatively in prokaryotic host cells. The proteins of the presentinvention could be endogenous prokaryotic proteins or aheterologous/eukaryotic proteins. In what follows, non-limiting examplesof different applications of the invention are provided.

Another aspect of the present invention relates to a method ofidentifying a candidate protein which binds a target protein. Thismethod includes providing a first expression system comprising a nucleicacid molecule encoding a first fragment of a reporter protein molecule,a nucleic acid molecule encoding first signal sequence, and a nucleicacid molecule encoding a target protein, where the nucleic acid moleculeencoding the first fragment, the nucleic acid molecule encoding thefirst signal sequence, and the nucleic acid molecule encoding the targetprotein are operatively coupled to permit their expression in aprokaryotic host cell as a first fusion protein. This method alsoincludes providing a second expression system comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a candidate protein, wherein the nucleic acidmolecule encoding the second fragment, the nucleic acid moleculeencoding the second signal sequence, and the nucleic acid moleculeencoding the candidate protein are operatively coupled to permit theirexpression in a prokaryotic host cell as a second fusion protein. Thismethod further includes transforming a prokaryotic host cell with thefirst expression system and the second expression system, culturing thetransformed prokaryotic host cell under conditions effective to expressthe first and the second fusion proteins and to transport the firstfusion protein and the second fusion protein to the prokaryotic hostcell's periplasm, with at least one of the first fusion protein or thesecond fusion protein being co-translationally transported to theperiplasm. The prokaryotic host cells with reporter protein moleculeactivity are detected as those where binding between the candidateprotein and the target protein has occurred. The candidate protein isidentified as having the ability to bind to the target protein based onwhether the host cell has reporter protein activity.

The proteins that can be used as target proteins and candidate proteinsare mentioned supra.

The reporter systems of the present invention can be used in manyapplications such as in human therapeutics, diagnostics, andprognostics, in high-throughput screening systems for the discovery andvalidation of pharmaceutical targets and drugs, as well as discovery ofgenes that modulate protein interactions. Massive parallel mapping ofpair-wise protein-protein interactions within and between the proteomesof cells, tissues, and pathogenic organisms, selection of antibodyfragments or other binding proteins to whole proteomes, antigenidentification for antibodies, epitope identification for antibodies,high-throughput screens for inhibitors of any protein-proteininteraction can be done using the methods of the present invention. Inone embodiment, the reporter system can be combined with directedevolution methods and used to engineer ultra-high affinity interactionsbetween proteins such as antibody-antigen pairs.

By combining the methods and compositions of the invention withstate-of-the-art methods for construction of high-titer, high-complexitycDNA libraries, it will be possible to identify interaction partners ofa specific test protein from, for example, mammalian cells (i.e.,perform functional genomics at the protein level). For this application,cDNA libraries can be constructed wherein the cDNA coding sequence isfused to a sequence encoding the reporter protein fragments of thepresent invention. A sequence encoding a binding protein of interestwill be fused to a reporter protein fragment in a first vector. In asecond series of vectors, a second reporter protein fragment will befused to a variety of different proteins that will be tested for theirability to bind to the protein of interest. Testing will be conducted byco-transformation of prokaryotic host cells with the first and one ofthe series of second vectors. Those test proteins which are capable ofbinding to the protein of interest will allow detection of a reportersignal in cells in which they are co-expressed with the protein ofinterest.

This aspect of the present invention, the method can be separatelycarried out with a plurality of second expression systems containing aplurality of different nucleic acid molecules encoding different secondproteins. This plurality of different second proteins can be encoded bymembers of a cDNA library. The methods of the present invention, couldbe adapted for efficient simultaneous detection of multitudes ofinteractions among proteins within cells, including expressed sequencelibraries, cDNA libraries, single-chain antibody fragment (scFv)libraries, and scaffolded peptide libraries. They could also be used forrapid selection of binding molecules from single-chain antibody fragment(scFv) libraries, or from scaffolded peptide libraries for use asreagents in functional genomics studies, or for identification ofnatural ligands and epitopes by homology. Target interactions identifiedusing the present invention, could be used immediately to screen forcandidate compounds that act as inhibitors or activators of theprotein-protein interaction.

The methods disclosed herein enable the detection and quantitation ofbinding events in cell lysates, as well as in intact prokaryotic hostcells. The detectable signal is produced by the complementing reporterfragments and can serve as an indicator of binding between the putativebinding pair (or target and candidate protein), either directly orindirectly via a third substance. Signals from the complementingreporter fragments could be detected by methods which include, forexample, light emission and absorbance, genetic selection such asantibiotic selection, immunological techniques, such asimmunofluorescent labeling. Exemplary signals include chromogenic,fluorescent and luminescent signals. These signals can be detected andquantitated visually or through the use of spectrophotometers,fluorimeters, microscopes, scintillation counters or otherinstrumentation known in the art. Assay solutions can be designed anddeveloped for a particular system. The reporter systems disclosed hereincan be used to conduct assays in systems, such as buffered cell freeextracts of host cells, cell interiors, solutions of cells, solutions ofcell lysates, and solutions of cell fractions, such as nuclearfractions, cytoplasmic fractions, and membrane fractions. Methods forpreparing assay solutions, such as enzyme assay solutions, cellextracts, and cell suspensions, known in the art may be used. Forexample, physiologically compatible buffers such as phosphate bufferedsaline may be used.

In one embodiment of this aspect of the invention, the reporter proteinmolecule activity can be quantitated. In a preferred embodiment, thereporter protein activity detected among various candidate proteins iscompared and used to identify the strongest binding candidate protein.This comparison among the plurality of candidate proteins can also beused to determine which of a plurality of candidate proteins bind to thetarget protein, or to rank the candidate proteins according to theirbinding affinities. The reporter protein typically will have a uniqueenzymatic activity or structure that enables it to be distinguished fromother proteins present in the prokaryotic host cell or lysate. Themethods of quantification of a reporter protein activity are well knownin the art. The activity of the transcribed reporter protein, orquantification of the activity of the reporter protein, provides anindirect measurement of binding between the putative members of thebinding pair. Reporter assays enable the identification of bindingaffinity and factors that control binding between the putative membersof the binding pair. Uses for reporter activity assay include, forexample, identification of factors that control or influenceprotein-protein interactions, study of mechanisms that influence proteininteractions, screening of candidate compounds or candidate genes thatinfluence protein-protein interactions.

In reporter protein assays with poor sensitivity, it is difficult todistinguish negative results caused by the lack of expression orlow-level assay sensitivity. This problem can be overcome with assays ofgreater sensitivity. Multiple assays are commonly used to providecontrols for efficiency of transfection. In such assays, cells aretransfected with a mixture of two separate plasmids, each having areporter protein molecule controlled by different regulatory sequences.The expression of one reporter protein is controlled by differentregulatory regions being studied while the other reporter gene, actingas a control, is generally constitutively expressed by a standardpromoter or enhancer. The activity of the experimental reporter enzymeis normalized to the activity of the control reporter enzyme. Similarly,to understand the effects of a candidate gene or a candidate drug onprotein-protein interaction, control experiments can be run in theabsence of the candidate compound or gene and used to normalize dataobtained in the presence of the candidate compound or gene. To providerelevant experimental information, reporter assays must be sensitive,thus enabling the detection of low levels of reporter protein in celllines that transfect poorly. The sensitivity of a reporter gene assay isa function of the detection method as well as reporter mRNA and proteinturnover, and endogenous (background) levels of the reporter activity.

Commonly used detection techniques use isotopic, calorimetric,fluorometric or luminescent enzyme substrates and immunoassay-basedprocedures with isotopic or color endpoints. Many of these systems,however, have disadvantages that limit their usefulness in these assays.For example, isotopic substrates and immunoassays are limited by thecost, sensitivity and inconvenience of using radioisotopes. Fluorometricsystems require external light sources that must be filtered todiscriminate fluorescent signal, thereby limiting the sensitivity andincreasing complexity of the detection system. Furthermore, fluorescencefrom endogenous source can interfere with fluorometric measurements.Colorimetric systems lack the sensitivity desired for sensitive reportergene assays. Chemiluminescent and bioluminescent assays, on the otherhand, have been found to be more rapid and sensitive than colorimetricassays and fluorometric assays.

This application will also be useful in screening for agonists andantagonists of medically-relevant protein interactions. The assays andmethods of the invention can also be carried out in the presence ofextracellular signaling molecules, growth factors or differentiationfactors, activated or inactivated genes or signals, peptides, organiccompounds, drugs or synthetic analogs, or the like, whose presence oreffects might alter the potential for interaction between the targetprotein and the candidate protein.

Another aspect of the present invention is related to a method ofidentifying a candidate gene which modulates the binding between a firstprotein and a second protein. This method includes providing a firstexpression system comprising a nucleic acid molecule encoding firstfragment of a reporter protein molecule, a nucleic acid moleculeencoding a first signal sequence, and a nucleic acid molecule encoding afirst protein, where the nucleic acid molecule encoding the firstfragment, the nucleic acid molecule encoding the first signal sequence,and the nucleic acid molecule encoding the first protein are operativelycoupled to permit their expression in a prokaryotic host cell as a firstfusion protein. This method also includes providing a second expressionsystem for expressing the second protein comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second protein, where the nucleic acid moleculeencoding the second fragment, the nucleic acid molecule encoding thesecond signal sequence, and the nucleic acid molecule encoding thesecond protein are operatively coupled to permit their expression in aprokaryotic host cell as a second fusion protein. This method furtherinvolves providing a candidate gene in a form suitable for expression ina prokaryotic host cell, transforming the prokaryotic host cell with thefirst expression system, the second expression system, and the candidategene, and culturing the transformed prokaryotic host cell underconditions effective to, in the absence of the candidate gene, expressthe first fusion protein, the second fusion protein, and the proteinencoded by the candidate gene and transport the first fusion protein andthe second fusion protein to the prokaryotic host cell's periplasm withat least one of the first fusion protein or the second fusion proteinbeing co-translationally transported to the periplasm. Any reporteractivity in the transformed prokaryotic host cell is detected, andprokaryotic host cells, with reporter activity that is different thanthat achieved without transformation of the candidate gene areidentified, as containing a candidate gene which modulates bindingbetween the first and second proteins.

The proteins that can be used as the first protein and the secondprotein are mentioned supra.

In one embodiment of this aspect of the invention, the presence of thecandidate gene decreases the reporter activity and is identified as acandidate gene whose presence inhibits the binding between the firstprotein and the second protein. In another embodiment the presence ofthe candidate gene increases the reporter activity and is identified asactivating the binding between the first protein and the second protein.

In another embodiment, the method is separately carried out with aplurality of second expression systems containing a plurality ofdifferent nucleic acid molecules encoding different second proteins,such that the second proteins are encoded by members of a cDNA library.A library of all the protein members of a proteome of interest can beconstructed and used. Libraries derived from nucleotide sequences thatcontain all members of a total protein population (i.e. a proteome) ofinterest may be isolated from a host cell such as a prokaryotic or aeukaryotic cell, or also from a viral host. Viral hosts that encode foroncogenes are of particular interest. Mammalian tumor cells, immunecells and endothelial cells also provide proteomes of particularinterest for the present invention.

In one embodiment of this aspect of the invention, the reporter proteinactivity is quantitated as described supra. In a preferred embodiment,the reporter protein activity detected among various candidate genes iscompared and used to identify the strongest modulator of the interactionbetween the first protein and the second protein. The candidate gene canmodulate the protein-protein interaction in a way such that it leads toactivation the interaction or it leads to the inhibition of theinteraction.

Another aspect of the present invention is related to a method ofidentifying a candidate compound which modulates binding between a firstprotein and a second protein. This method includes providing a firstexpression system comprising a nucleic acid molecule encoding firstfragment of a reporter protein molecule, a nucleic acid moleculeencoding a first signal sequence, and a nucleic acid molecule encoding afirst protein, where the nucleic acid molecule encoding the firstfragment, the nucleic acid molecule encoding the first signal sequence,and the nucleic acid molecule encoding the first protein are operativelycoupled to permit their expression in a prokaryotic host cell as a firstfusion protein. This method also includes providing a second expressionsystem for expressing the second protein comprising a nucleic acidmolecule encoding a second fragment of the reporter protein molecule, anucleic acid molecule encoding a second signal sequence, and a nucleicacid molecule encoding a second protein, where the nucleic acid encodingthe second fragment, the nucleic acid encoding the second signalsequence, and the nucleic acid encoding the second protein areoperatively coupled to permit their expression in a prokaryotic hostcell as a second fusion protein. This method further involves providinga candidate compound, transforming a host prokaryotic cell with thefirst expression system and the second expression system, and culturingthe transformed host prokaryotic cell under conditions effective toexpress the first and the second fusion proteins and transport the firstfusion protein and the second fusion protein to the prokaryotic hostcell's periplasm with at least one of the first fusion protein or thesecond fusion protein being co-translationally transported to theperiplasm. The candidate compound is contacted with the culturedprokaryotic host cell. The reporter activity is detected in thetransformed prokaryotic host cell, and candidate compounds contactingthe prokaryotic host cells, with reporter activity that is differentthan that achieved in transformed prokaryotic host cells not contactedwith the candidate compound are identified as modulating binding betweenthe first protein and the second protein.

In one embodiment, the candidate compound decreases reporter activityand is identified as inhibiting binding between the first protein andthe second protein. In the same manner a candidate compound, whosepresence increases reporter activity is identified as activating bindingbetween the first protein and the second protein. In another aspect ofthe present invention a plurality of candidate compounds could be usedand screened for their effect on the protein-protein interaction.

EXAMPLES Example 1 Construction of Vectors

All cloning was performed using standard molecular biologicaltechniques. A gene sequence comprising a (GGGGS)₃-NGR linker sequencefollowed by residues 198-286 of TEM-1 BLA was cloned between the BamHIand HindIII sites of pDMB to create pDMB-ωBLA. Genes to be fused to theωBLA fragment were then cloned into this vector between XbaI and SalIsites. Genes to be fused to the αBLA fragment were cloned between theKpnI and BamHI sites of vector αGS-Jun (Wehrman, T., et al.,“Protein-protein Interactions Monitored in Mammalian Cells ViaComplementation of Beta-lactamase Enzyme Fragments,” Proc Natl Acad SciUSA 99:3469-3474 (2002), which is hereby incorporated by reference inits entirety). Fos and Jun leucine knockouts and scFv-GCN4 were as in(DeLisa, M. P., “Versatile Selection Technology for IntracellularProtein-protein Interactions Mediated by Bacterial Hitchhiker TransportMechanism,”Proc Natl Acad Sci USA 106:3692-3697 (2009), which is herebyincorporated by reference in its entirety). Template DNA for scFvs D10,and gpd was kindly provided by Andreas Plückthun.

Example 2 Expression of Fusion Proteins and Cell Growth Assays

E. coli MC4100 cells were co-transformed with plasmids pDMB-P1-ωBLA(CmR) and aGS-P2 (KanR), where P1 and P2 represent proteins of interest.Cells were grown overnight at 37° C. in LB medium containing 20 μg/mLchloramphenicol (Cm) and 50 μg/mL kanamycin (Kan). Screening of cells onLB agar was performed by first normalizing overnight cultures by OD₆₀₀and then spotting 5 μL, of serially diluted (10-10⁶-fold) cells on LBagar plates with 12, 25, 50, or 50 μg/mL of ampicillin (Amp). In allcases, the plates were incubated 16 h at 37° C. and then imaged using aChemiDoc System (BioRad). For MBC/MIC determination, approximately 200colony forming units (CFUs) of each clone were plated on LB agar platescontaining 0, 3, 6, 12, 25, 50, 100, 200, 400, 800, or 1600 μg/mL Amp or20 μg/mL Cm. The minimum bacteriocidal concentration was determined asthe minimum Amp concentration at which no colonies appeared on theplates. Minimum inhibitory concentration (MIC) was determined as theminimum concentration of Amp on which colony size was significantlysmaller than control.

It was hypothesized that detecting protein interactions in the periplasmcan be used to engineer antibody fragments with in vivo activity in theperiplasm. The periplasm is ideal for antibody engineering as itsoxidizing environment allows for the formation of disulfide bonds, whichmany antibody fragments require to function (Auf der Maur, A., et al.,“Antigen-independent Selection of Stable Intracellular Single-chainAntibodies,” FEBS Lett 508:407-12 (2001), which is hereby incorporatedby reference in its entirety). The approach used in the presentinvention was to fuse known interacting protein domains to genesencoding two split β-lactamase fragments using a two-plasmid system(Wehrman, T., et al., “Protein-protein Interactions Monitored inMammalian Cells Via Complementation of Beta-lactamase Enzyme Fragments,”Proc Natl Acad Sci USA 99:3469-3474 (2002), which is hereby incorporatedby reference in its entirety)(see FIG. 2A). The α fragment encodesresidues 1-196 of TEM-1 Bla, while the ω fragment encodes residues197-286 out of 286. The α fragment contains the native signal sequenceof Bla (residues 1-23), which targets the C-terminally fused protein tothe Sec translocon for transport into the periplasm. In the construct ofthe present invention, the protein of interest fused to the ω fragmentis targeted to the SRP pathway for co-translational translocation.

Example 3 Antibody-Antigen Interactions are Reported in Two Cases

To determine the ability of the assay of the present invention to detectand eventually engineer antibody-antigen interactions, interactingprotein pairs as well as non-interacting proteins were cloned into twoplasmids carrying the two inactive Bla fragments (FIG. 2A). In thisstudy, two antibody-antigen pairs were examined. First, an scFv which isknown to bind the GCN4 leucine zipper was tested. In addition to thescFv and wild-type GCN4 pair, a double proline mutant, GCN4-PP as anantigen for the scFv was also tested. GCN4 is normally a homodimer, buta double proline mutant of GCN4 (GCN4-pp) exhibits reduced dimerization,thereby reducing the amount of inactive α/α dimers in the assay. FIGS.2B-D show the results of spot plating of the interacting pairs, withα-Jun as a negative control. The spot plates correspond to a 16-foldhigher MBC (minimum bacteriocidal concentration, see Example 2 for fulldefinition of MIC/MBC) on ampicillin for GCN4-PP and a 2-fold higher MBCfor the GCN4 wild-type (homodimer). A difference of this magnitude ispromising for future library selection processes.

In the case of GCN4, an antibody fragment that binds a leucine zipper, ashort peptide fragment was already tested. Since a goal of this assay isengineering protein-protein interactions, an antibody fragment with aprotein antigen was tested. D10, an scFv with affinity for the phageprotein gpD, an 11.4 kDa capsid protein from bacteriophage lambda wasused (Koch, H., et al., “Direct Selection of Antibodies from ComplexLibraries with the Protein Fragment Complementation Assay,” J Mol Biol357:427-441 (2006), which is hereby incorporated by reference in itsentirety). Indeed, the results with D10 were similar (spot plating shownin FIGS. 2B-D), with an MBC roughly 8-fold higher than the Jun negativecontrol and 16-fold higher than the GCN4-PP negative control.

Example 4 Leucine Zipper Interactions are Reported Reliably

The hydrophobic interaction between Fos and Jun leucine zippers is welldocumented and these proteins are often used as standards for proteininteraction (Wehrman, T., et al., “Protein-protein InteractionsMonitored in Mammalian Cells via Complementation of Beta-lactamaseEnzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002), which ishereby incorporated by reference in its entirety). The K_(D) of theirinteraction has been reported in the nanomolar range (Pernelle, C., etal., “An Efficient Screening Assay for the Rapid and PreciseDetermination of Affinities between Leucine Zipper Domains,”Biochemistry 32:11682-11687 (1993), which is hereby incorporated byreference in its entirety). In the present study, Fos was fused to the ωfragment and Jun to the α fragment of β-lactamase (Bla). As a negativecontrol, a potential binding partner of the leucine zipper GCN4 wasincorporated, which does not bind Fos. When placed in the PCA, Fos andJun were found to have ampicillin activity well above that of thenegative control (FIG. 2C). After successful spot plating, theinteractions were further characterized by constructing a series ofpoint mutants that have been shown to have lower affinity (due toknockout of leucine residues). The MIC/MBC of these interactions andspot plating of these mutants can be found in FIG. 3. It is notable thatthe assay is apparently able to correlate Bla activity with levels ofinteraction along a spectrum of affinities.

It is shown that antibody-antigen reactions show a large difference inMIC/MBC from non-specific interactions. This can be used to selectinteracting pairs from a library of antibody fragments. The Tomlinsonlibraries, libraries of scFv antibody fragments with randomizedcomplementarity determining regions, provide huge diversity. Normally,these libraries are used to isolate binding by phage display. Thepresent invention is also related to cloning such libraries into thereporter system of the present invention, genetically fusing the scFvsto the ω fragment of Bla and selecting for interactions with a desirableantigen. In order to provide a well-folded, soluble antigen, the E. colimaltose binding protein can be used as a potential binding substrate.After cloning the library, 2 plasmids can be co-transformed: (1) scFvlibrary-ω-Bla and (2) α-Bla-MalE, then selected for growth on ampicillinplates. The resulting hits can be analyzed by surface plasmon resonance(BIACore) to determine dissociation constants and further evolved viathe creation of error-prone PCR libraries (with a target error rate of3-4 mutations per gene) and selection on increasing ampicillinconcentration media. Such a study will serve two purposes: (1) theselection of a novel antibody-antigen interaction and (2) the study ofenrichment of affinity and its effect on Bla activity. The presentinvention has demonstrated that Bla activity can change with affinityand increasing affinity will provide a chance not only to track thecorrelation of ampicillin activity with affinity, but also the upper andlower bounds of affinity capable of detection in this assay. Anadditional use for this assay is selection for interactions of proteinantigens with full-length IgGs, produced in the periplasm fused to a Blafragment, then selected in the manner above.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A reporter system for detection of protein-proteininteractions in the periplasm of a prokaryotic host cell, said systemcomprising: a first expression system comprising: a nucleic acidmolecule encoding a first fragment of a reporter protein molecule; anucleic acid molecule encoding a first signal sequence directed to asignal recognition particle (SRP) transport pathway; and a nucleic acidmolecule encoding a first member of a putative binding pair, whereinsaid nucleic acid molecule encoding the first fragment, said nucleicacid molecule encoding the first signal sequence, and said nucleic acidmolecule encoding the first member of the putative binding pair areoperatively coupled to permit their expression in a prokaryotic hostcell as a first fusion protein and a second expression systemcomprising: a nucleic acid molecule encoding a second fragment of thereporter protein molecule; a nucleic acid molecule encoding a secondsignal sequence; and a nucleic acid molecule encoding a second member ofthe putative binding pair, wherein said nucleic acid molecule encodingthe second fragment, said nucleic acid molecule encoding the secondsignal sequence, and said nucleic acid molecule encoding the secondmember of the putative binding pair are operatively coupled to permittheir expression in a prokaryotic host cell as a second fusion protein,wherein the first signal sequence and optionally the second signalsequence are capable of directing co-translational transport of at leastone of the fusion proteins, when expressed in a prokaryotic host cell,into the host cell periplasm where, when present, the first and secondmembers of the putative binding pair bind together and the first andsecond fragments of the reporter protein molecule are reconstituted,thereby producing an active reporter protein.
 2. The reporter systemaccording to claim 1, wherein both the first signal sequence and thesecond signal sequence are capable of directing co-translationaltransport of the first and the second fusion proteins to the periplasm.3. The reporter system according to claim 1, wherein the first member ofthe putative binding pair and the second member of the putative bindingpair have a binding affinity which is stronger than the binding affinityof the first fragment and the second of the reporter protein.
 4. Thereporter system according to claim 1, wherein said reporter proteinmolecule is selected from the group consisting of a monomeric protein, amultimeric protein, a monomeric receptor, a multimeric receptor, amultimeric biomolecular complex, adenylate cyclase, alkalinephosphatase, β-lactamase, cellulase, chloramphenicol acetyl transferase(CAT), disulfide bond oxidase A (DsbA), maltose binding protein (MBP),methyltransferase, dihydrofolate reductase (DHFR), luciferase,thymidylate synthase, thymidine kinase, Trp1N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and fluorescentproteins.
 5. The reporter system according to claim 4, wherein thereporter protein molecule is β-lactamase.
 6. The reporter proteinmolecule according to claim 5, wherein the first fragment and the secondfragment of the reporter protein molecule, respectively, compriseβ-lactamase's α fragment having the amino acid sequence of SEQ ID NO: 2,and β lactamase's ω fragment having the amino acid sequence of SEQ IDNO:
 3. 7. The reporter system according to claim 1, wherein the secondsignal sequence is directed to a transport pathway selected from thegroup consisting of Sec pathway, SRP pathway, and Tat pathway.
 8. Thereporter system according to claim 7, wherein the first signal sequenceand the second signal sequences are directed to the SRP pathway and areselected from the group consisting ssArtI, ssDsbA, ssFlgA, ssLivJ,ssSfmC, ssSTII, ssTolB, ssTorT, ssYraP, and ssYraI.
 9. The reportersystem according to claim 7, wherein the second signal sequence isdirected to the Sec pathway and is selected from the group consistingssAppA, ssBla, ssClyA, ssLep, ssMalE, ssOmpA, ssOmpT, ssOmpX, ssPelB(Erwinia chrysanthemi), ssPhoA, ssRbsB, and ssYebF.
 10. The reportersystem according to claim 7, wherein the second signal sequence isdirected to the Tat pathway and is selected from the group consistingssFdnG, ssFdoG, ssNapG, ssNrfC, ssHyaA, ssYnfE, ssWcaM, ssTorA, ssNapA,ssYagT, ssYcbK, ssDmsA, ssYdhX, ssYahJ, ssYedY, ssCueO, ssSufI, ssYcdB,ssTorZ, ssHybA, ssYnfF, ssHybO, ssAmiC, ssAmiA, ssYfhG, ssMdoD, ssFhuD,ssYaeI, and ssYcdO.
 11. The reporter system according to claim 1,wherein the prokaryotic host cell is a gram negative bacteria.
 12. Thereporter system according to claim 1, wherein the putative binding pairis selected from the group of proteins consisting a membraneprotein-soluble binding protein pair, a membrane protein-membraneprotein binding pair, a biotin-avidin binding pair, ligand-receptorbinding pair, and an antibody-antigen pair.
 13. The reporter systemaccording to claim 12, wherein the putative binding pair is anantibody-antigen pair, wherein the antibody is selected from the groupconsisting of monoclonal antibody, bispecific antibody, single-chainantibody (scAb), single-chain Fv fragment (scFv), scFv₂, dsFv, scFv-Fc,Fab, F(ab′)₂, F(ab′)₃, V_(L), diabody, single domain antibody, camelidantibody, triabody, tetrabody, minibody, one-armed antibody, andimmunoglobulin (Ig), IgM, IgE, IgA, IgD, IgG, IgG-ΔC_(H)2, and whereinthe antigen is selected from the group consisting of cell surfacereceptors, proteins regulating apoptosis, proteins that regulateprogression of the cell-cycle, proteins involved in the development oftumors, transcriptional-regulatory proteins, translation regulatoryproteins, proteins that affect cell interactions, cell adhesionmolecules, proteins which are members of ligand-receptor pairs, proteinsthat participate in the folding of other proteins, SNARE protein family,and proteins involved in targeting to intracellular compartments. 14.The reporter system according to claim 12, wherein the putative bindingpair is a receptor-ligand pair, wherein the receptor is selected fromthe group consisting of Fc receptors (FcR), single-chain MHC, andsingle-chain T-cell receptor (sc-TCR).
 15. The reporter system accordingto claim 12, wherein the putative binding pair is a membraneprotein-membrane protein binding pair or membrane protein-solubleprotein binding pair, wherein the membrane protein is selected from thegroup consisting of monotopic membrane proteins, polytopic membraneproteins, transmembrane proteins, G protein-coupled receptors (GPCRs),ion channels, SNARE protein family, integrin adhesion receptor, andmulti-drug efflux transporters.
 16. A method of identifying a candidateprotein which binds a target protein, said method comprising: a)providing a first expression system comprising: a nucleic acid moleculeencoding a first fragment of a reporter protein molecule; a nucleic acidmolecule encoding first signal sequence directed to a signal recognitionparticle (SRP) transport pathway; and a nucleic acid molecule encoding atarget protein, wherein said nucleic acid molecule encoding the firstfragment, said nucleic acid molecule encoding the first signal sequence,and said nucleic acid molecule encoding the target protein areoperatively coupled to permit their expression in a prokaryotic hostcell as a first fusion protein and b) providing a second expressionsystem comprising: a nucleic acid molecule encoding a second fragment ofthe reporter protein molecule; a nucleic acid molecule encoding a secondsignal sequence; and a nucleic acid molecule encoding a candidateprotein, wherein said nucleic acid molecule encoding the secondfragment, said nucleic acid molecule encoding the second signalsequence, and said nucleic acid molecule encoding the candidate proteinare operatively coupled to permit their expression in a prokaryotic hostcell as a second fusion protein; c) transforming a prokaryotic host cellwith said first expression system and said second expression system; d)culturing the transformed prokaryotic host cell under conditionseffective to express the first and the second fusion proteins and totransport the first fusion protein and the second fusion protein to theprokaryotic host cell's periplasm, wherein the first signal sequence andoptionally the second signal sequence are capable of directingco-translational transport of at least one of the fusion proteins, whenexpressed in a prokaryotic host cell, into the host cell periplasm; e)detecting the prokaryotic host cells with reporter protein moleculeactivity as those where binding between the candidate protein and thetarget protein has occurred; and f) identifying the candidate protein ashaving the ability to bind to the target protein based on whether thehost cell has reporter protein activity.
 17. The method according toclaim 16, wherein the target and candidate proteins are selected fromthe group of proteins consisting of a membrane protein-soluble bindingprotein pair, a membrane protein-membrane protein binding pair, abiotin-avidin binding pair, ligand-receptor binding pair, and anantibody-antigen pair.
 18. The method according to claim 16, whereinsaid reporter protein molecule is selected from the group consisting ofa monomeric protein, a multimeric protein, a monomeric receptor, amultimeric receptor, a multimeric biomolecular complex, adenylatecyclase, alkaline phosphatase, β-lactamase, cellulase, chloramphenicolacetyl transferase (CAT), disulfide bond oxidase A (DsbA), maltosebinding protein (MBP), methyltransferase, dihydrofolate reductase(DHFR), luciferase, thymidylate synthase, thymidine kinase, Trp1N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and fluorescentproteins.
 19. The method according to claim 16, wherein the secondsignal sequence is directed to a transport pathway selected from thegroup consisting of Sec pathway, SRP pathway, and Tat pathway.
 20. Themethod according to claim 16, wherein the prokaryotic host cell is agram negative bacteria.
 21. The method according to claim 16 whereinsaid detecting is carried out quantitatively.
 22. The method accordingto claim 21 further comprising: comparing the quantitative reporterprotein activity detected among various candidate proteins to identifythe strongest binding candidate protein.
 23. The method according toclaim 16, wherein said method is carried out to determine which of aplurality of candidate proteins bind to the target protein.
 24. A methodof identifying a candidate gene which modulates binding between a firstprotein and a second protein, said method comprising: a) providing afirst expression system comprising a nucleic acid molecule encodingfirst fragment of a reporter protein molecule; a nucleic acid moleculeencoding a first signal sequence directed to a signal recognitionparticle (SRP) transport pathway; and a nucleic acid molecule encoding afirst protein, wherein said nucleic acid molecule encoding the firstfragment, said nucleic acid molecule encoding the first signal sequence,and said nucleic acid molecule encoding the first protein areoperatively coupled to permit their expression in a prokaryotic hostcell as a first fusion protein; b) providing a second expression systemfor expressing the second protein comprising: a nucleic acid moleculeencoding a second fragment of the reporter protein molecule; a nucleicacid molecule encoding a second signal sequence; and a nucleic acidmolecule encoding a second protein, wherein said nucleic acid moleculeencoding the second fragment, said nucleic acid molecule encoding thesecond signal sequence, and said nucleic acid molecule encoding thesecond protein are operatively coupled to permit their expression in aprokaryotic host cell as a second fusion protein; c) providing acandidate gene in a form suitable for expression in a prokaryotic hostcell; d) transforming the prokaryotic host cell with the firstexpression system, the second expression system, and the candidate gene;e) culturing the transformed prokaryotic host cell under conditionseffective to, in the absence of the candidate gene, express the firstfusion protein, the second fusion protein, and the protein encoded bythe candidate gene and transport the first fusion protein and the secondfusion protein to the prokaryotic host cell's periplasm, wherein thefirst signal sequence and optionally the second signal sequence arecapable of directing co-translational transport of at least one of thefusion proteins, when expressed in a prokaryotic host cell, into thehost cell periplasm; f) detecting any reporter activity in thetransformed prokaryotic host cell; and g) identifying prokaryotic hostcells, with reporter activity that is different than that achievedwithout transformation of the candidate gene, as containing a candidategene which modulates binding between the first and second proteins. 25.The method according to claim 24, wherein a candidate gene, whosepresence decreases reporter activity is identified as inhibiting bindingbetween the first protein and the second protein.
 26. The methodaccording to claim 24, wherein a candidate gene, whose presenceincreases reporter activity is identified as activating binding betweenthe first protein and the second protein.
 27. The method according toclaim 24, wherein said method is separately carried out with a pluralityof second expression systems containing a plurality of different nucleicacid molecules encoding different second proteins, wherein the secondproteins are encoded by members of a cDNA library.
 28. The methodaccording to claim 24, wherein the first protein and the second proteinhave a binding affinity which is stronger than the binding affinity ofthe first fragment and the second fragment of the reporter protein. 29.The method according to claim 24, wherein said reporter protein moleculeis selected from the group consisting of a monomeric protein, amultimeric protein, a monomeric receptor, a multimeric receptor, amultimeric biomolecular complex, adenylate cyclase, alkalinephosphatase, β-lactamase, cellulase, chloramphenicol acetyl transferase(CAT), disulfide bond oxidase A (DsbA), maltose binding protein (MBP),methyltransferase, dihydrofolate reductase (DHFR), luciferase,thymidylate synthase, thymidine kinase, Trp1N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and fluorescentproteins.
 30. The method according to claim 24, wherein the secondsignal sequence is directed to a transport pathway selected from thegroup consisting of Sec pathway, SRP pathway, and Tat pathway.
 31. Themethod according to claim 24, wherein the prokaryotic host cell is agram negative bacteria.
 32. The method according to claim 24, whereinthe first and the second protein are selected from the group of proteinsconsisting of a membrane protein-soluble binding protein pair, amembrane protein-membrane protein binding pair, a biotin-avidin bindingpair, ligand-receptor binding pair, and an antibody-antigen pair. 33.The method according to claim 24, wherein said detecting is carried outquantitatively.
 34. The method according to claim 33 further comprising:comparing the quantitative reporter protein activity detected amongvarious candidate genes to identify the candidate gene which is thestrongest modulator of the interaction between the first protein and thesecond protein.
 35. A method of identifying a candidate compound whichmodulates binding between a first protein and a second protein, saidmethod comprising: a) providing a first expression system comprising anucleic acid molecule encoding first fragment of a reporter proteinmolecule; a nucleic acid molecule encoding a first signal sequencedirected to a signal recognition particle (SRP) transport pathway; and anucleic acid molecule encoding a first protein, wherein said nucleicacid molecule encoding the first fragment, said nucleic acid moleculeencoding the first signal sequence, and said nucleic acid moleculeencoding the first protein are operatively coupled to permit theirexpression in a prokaryotic host cell as a first fusion protein b)providing a second expression system for expressing the second proteincomprising: a nucleic acid molecule encoding a second fragment of thereporter protein molecule; a nucleic acid molecule encoding a secondsignal sequence; and a nucleic acid molecule encoding a second protein,wherein said nucleic acid encoding the second fragment, said nucleicacid encoding the second signal sequence, and said nucleic acid encodingthe second protein are operatively coupled to permit their expression ina prokaryotic host cell as a second fusion protein; c) providing acandidate compound; d) transforming a host prokaryotic cell with thefirst expression system and the second expression system; e) culturingthe transformed host prokaryotic cell under conditions effective toexpress the first and the second fusion proteins and transport the firstfusion protein and the second fusion protein to the prokaryotic hostcell's periplasm, wherein the first signal sequence and optionally thesecond signal sequence are capable of directing co-translationaltransport of at least one of the fusion proteins, when expressed in aprokaryotic host cell, into the host cell periplasm; f) contacting thecandidate compound with the cultured prokaryotic host cell; g) detectingreporter activity in the transformed prokaryotic host cell; and h)identifying candidate compounds contacting the prokaryotic host cells,with reporter activity that is different than that achieved intransformed prokaryotic host cells not contacted with the candidatecompound, as modulating binding between the first protein and the secondprotein.
 36. The method according to claim 35, wherein the candidatecompound, whose presence decreases reporter activity is identified asinhibiting binding between the first protein and the second protein. 37.The method according to claim 35, wherein a candidate compound, whosepresence increases reporter activity is identified as activating bindingbetween the first protein and the second protein.
 38. The methodaccording to claim 35, wherein the binding affinity of the first proteinand the second protein is stronger than the binding affinity of thefirst and second fragments of the reporter protein.
 39. The methodaccording to claim 35, wherein said reporter protein molecule isselected from a group consisting of a monomeric protein, a multimericprotein, a monomeric receptor, a multimeric receptor, a multimericbiomolecular complex, adenylate cyclase, alkaline phosphatase,β-lactamase, cellulase, chloramphenicol acetyl transferase (CAT),disulfide bond oxidase A (DsbA), maltose binding protein (MBP),methyltransferase, dihydrofolate reductase (DHFR), luciferase,thymidylate synthase, thymidine kinase, Trp1N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and fluorescentproteins.
 40. The method according to claim 37, wherein the secondsignal sequence is directed to a transport pathway selected from thegroup consisting of Sec pathway, SRP pathway, and the Tat pathway. 41.The method according to claim 35, wherein the first and the secondprotein are selected from the group of proteins consisting of a membraneprotein-soluble binding protein pair, a membrane protein-membraneprotein binding pair, a biotin-avidin binding pair, ligand-receptorbinding pair, and an antibody-antigen pair.
 42. The method according toclaim 35, wherein the prokaryotic host cell is a gram negative bacteria.43. The method according to claim 35, wherein said detecting is carriedout quantitatively.
 44. The method according to claim 35 furthercomprising: comparing the quantitative reporter protein activitydetected among various candidate compounds to identify the compoundwhich is the strongest modulator of the interaction between the firstprotein and the second protein.
 45. The method according to claim 35,wherein the said method is carried out with a plurality of candidatecompounds.
 46. A reporter system for detection of protein-proteininteractions in the periplasm of a prokaryotic host cell, said systemcomprising: a first expression system comprising: a nucleic acidmolecule encoding an alpha (a) fragment of a β-lactamase reporterprotein molecule; a nucleic acid molecule encoding a first signalsequence directed to a signal recognition particle (SRP) transportpathway; and a nucleic acid molecule encoding a first member of aputative binding pair, wherein said nucleic acid molecule encoding thealpha (α) fragment, said nucleic acid molecule encoding the first signalsequence, and said nucleic acid molecule encoding the first member ofthe putative binding pair are operatively coupled to permit theirexpression in a prokaryotic host cell as a first fusion protein and asecond expression system comprising: a nucleic acid molecule encoding anomega (ω) fragment of the β-lactamase reporter protein molecule; anucleic acid molecule encoding a second signal sequence; and a nucleicacid molecule encoding a second member of the putative binding pair,wherein said nucleic acid molecule encoding the omega (ω) fragment, saidnucleic acid molecule encoding the second signal sequence, and saidnucleic acid molecule encoding the second member of the putative bindingpair are operatively coupled to permit their expression in a prokaryotichost cell as a second fusion protein, wherein the first signal sequenceand optionally the second signal sequence are capable of directingco-translational transport of at least one of the fusion proteins, whenexpressed in a prokaryotic host cell, into the host cell periplasmwhere, when present, the first and second members of the putativebinding pair bind together and alpha (α) and omega (ω) fragments of theβ-lactamase reporter protein molecule are reconstituted, therebyproducing an active reporter β-lactamase protein, wherein the bacterialhost cell is a gram negative bacteria.
 47. The reporter system accordingto claim 46, wherein both the first signal sequence and the secondsignal sequence are capable of directing co-translational transport ofthe first and the second fusion proteins to the periplasm.
 48. Thereporter system according to claim 46, wherein the first member of theputative binding pair and the second member of the putative binding pairhave a binding affinity which is stronger than the binding affinity ofthe alpha (α) fragment and the omega (ω) fragment of the reporterprotein.
 49. The reporter system according to claim 46, wherein thesecond signal sequence is directed to a transport pathway selected fromthe group consisting of Sec pathway, SRP pathway, and Tat pathway. 50.The reporter system according to claim 49, wherein the first signalsequence and the second signal sequences are directed to the SRP pathwayand are selected from the group consisting ssArtI, ssDsbA, ssFlgA,ssLivJ, ssSfmC, ssSTII, ssTolB, ssTorT, ssYraP, and ssYraI.
 51. Thereporter system according to claim 49, wherein the second signalsequence is directed to the Sec pathway and is selected from the groupconsisting ssAppA, ssBla, ssClyA, ssLep, ssMalE, ssOmpA, ssOmpT, ssOmpX,ssPelB (Erwinia chrysanthemi), ssPhoA, ssRbsB, and ssYebF.
 52. Thereporter system according to claim 49, wherein the second signalsequence is directed to the Tat pathway and is selected from the groupconsisting ssFdnG, ssFdoG, ssNapG, ssNrfC, ssHyaA, ssYnfE, ssWcaM,ssTorA, ssNapA, ssYagT, ssYcbK, ssDmsA, ssYdhX, ssYahJ, ssYedY, ssCueO,ssSufI, ssYcdB, ssTorZ, ssHybA, ssYnfF, ssHybO, ssAmiC, ssAmiA, ssYfhG,ssMdoD, ssFhuD, ssYaeI, and ssYcdO.
 53. The reporter system according toclaim 46, wherein the putative binding pair is selected from the groupof proteins consisting a membrane protein-soluble binding protein pair,a membrane protein-membrane protein binding pair, a biotin-avidinbinding pair, ligand-receptor binding pair, and an antibody-antigenpair.
 54. The reporter system according to claim 53, wherein theputative binding pair is an antibody-antigen pair, wherein the antibodyis selected from the group consisting of monoclonal antibody, bispecificantibody, single-chain antibody (scAb), single-chain Fv fragment (scFv),scFv₂, dsFv, scFv-Fc, Fab, F(ab′)₂, F(ab′)₃, V_(L), diabody, singledomain antibody, camelid antibody, triabody, tetrabody, minibody,one-armed antibody, and immunoglobulin (Ig), IgM, IgE, IgA, IgD, IgG,IgG-ΔC_(H)2, and wherein the antigen is selected from the groupconsisting of cell surface receptors, proteins regulating apoptosis,proteins that regulate progression of the cell-cycle, proteins involvedin the development of tumors, transcriptional-regulatory proteins,translation regulatory proteins, proteins that affect cell interactions,cell adhesion molecules, proteins which are members of ligand-receptorpairs, proteins that participate in the folding of other proteins, SNAREprotein family, and proteins involved in targeting to intracellularcompartments.
 55. The reporter system according to claim 53, wherein theputative binding pair is a receptor-ligand pair, wherein the receptor isselected from the group consisting of Fc receptors (FcR), single-chainMHC, and single-chain T-cell receptor (sc-TCR).
 56. The reporter systemaccording to claim 53, wherein the putative binding pair is a membraneprotein-membrane protein binding pair or membrane protein-solubleprotein binding pair, wherein the membrane protein is selected from thegroup consisting of monotopic membrane proteins, polytopic membraneproteins, transmembrane proteins, G protein-coupled receptors (GPCRs),ion channels, SNARE protein family, integrin adhesion receptor, andmulti-drug efflux transporters.